GK Domain Research Articles

Unraveling the evolutionary origin of the P5CS gene: a story of gene fusion and horizontal transfer.

The accumulation of proline in response to the most diverse types of stress is a widespread defense mechanism. In prokaryotes, fungi, and certain unicellular eukaryotes (green algae), the first two reactions of proline biosynthesis occur through two distinct enzymes, γ-glutamyl kinase (GK E.C. 2.7.2.11) and γ-glutamyl phosphate reductase (GPR E.C. 1.2.1.41), encoded by two different genes, ProB and ProA, respectively. Plants, animals, and a few unicellular eukaryotes carry out these reactions through a single bifunctional enzyme, the Δ1-pyrroline-5-carboxylate synthase (P5CS), which has the GK and GPR domains fused. To better understand the origin and diversification of the P5CS gene, we use a robust phylogenetic approach with a broad sampling of the P5CS, ProB and ProA genes, including species from all three domains of life. Our results suggest that the collected P5CS genes have arisen from a single fusion event between the ProA and ProB gene paralogs. A peculiar fusion event occurred in an ancestral eukaryotic lineage and was spread to other lineages through horizontal gene transfer. As for the diversification of this gene family, the phylogeny of the P5CS gene in plants shows that there have been multiple independent processes of duplication and loss of this gene, with the duplications being related to old polyploidy events.

The distal C terminus of the dihydropyridine receptor β1a subunit is essential for tetrad formation in skeletal muscle

The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.

Structural basis of dynamic P5CS filaments.

The bifunctional enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS) is vital to the synthesis of proline and ornithine, playing an essential role in human health and agriculture. Pathogenic mutations in the P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants. We have recently found that P5CS forms cytoophidia in vivo and filaments in vitro. However, it is difficult to appreciate the function of P5CS filamentation without precise structures. Using cryo-electron microscopy, here we solve the structures of Drosophila full-length P5CS in three states at resolution from 3.1 to 4.3 Å. We observe distinct ligand-binding states and conformational changes for the GK and GPR domains, respectively. Divergent helical filaments are assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations disturbing those interfaces prevent P5CS filamentation and greatly reduce the enzymatic activity. Our findings reveal that filamentation is crucial for the coordination between the GK and GPR domains, providing a structural basis for the catalytic function of P5CS filaments.

Crystal structure of the PDZ4 domain of MAGI2 in complex with PBM of ARMS reveals a canonical PDZ recognition mode

Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI2) is a neuronal scaffold protein that plays critical roles at synaptic junctions by assembling neurotransmitter receptors and cell adhesion proteins through its multiple protein-protein interaction domains, including six PDZ domains, two phosphoserine-phosphothreonine binding WW domains, and a guanylate kinase GK domain. Previous studies showed that MAGI2 participates in formation of tetrameric complexes with PDZ-GEF1, TrkA receptor, and ankyrin repeat-rich membrane spanning (ARMS) protein at late endosomes and is crucial for neurite outgrowth. However, the molecular mechanism governing the assembly of these complexes remains unknown. Here, we characterize the direct interaction between MAGI2 and ARMS through multiple biochemical assays. Moreover, our solved crystal structure of the truncated PDZ4/PBM (PDZ binding motifs) complex of MAGI2 and ARMS proteins (MAGI2-PDZ4/ARMS-PBM) reveals that the binding interface lies between the αB/βB groove from the PDZ4 of MAGI2 and the C-terminal PBM from ARMS. The structure reveals high similarity to others in this protein family where canonical PDZ/PBM interactions are observed. However, the conserved “GLGF” motif in the PSD-95-PDZ3 changes to “GFGF” in the MAGI2-PDZ4/ARMS-PBM complex. We further validated our crystal structure through serial mutagenesis assays. Taken together, our study provides the biochemical details and binding mechanisms that underpin the stabilization of the MAGI2-PDZ4/ARMS-PBM complex, thereby offering a biochemical and structural basis for further understanding of the functional roles of MAGI2, ARMS, PDZ-GEF1, and TrkA in forming the tetrameric receptor complex in neuronal signaling.

Entropy of stapled peptide inhibitors in free state is the major contributor to the improvement of binding affinity with the GK domain.

Stapled peptides are promising protein–protein interaction (PPI) inhibitors that can increase the binding potency. Different from small-molecule inhibitors in which the binding mainly depends on energetic interactions with their protein targets, the binding of stapled peptides has long been suggested to be benefited from entropy. However, it remains challenging to reveal the molecular features that lead to this entropy gain, which could originate from the stabilization of the stapled peptide in solution or from the increased flexibility of the complex upon binding. This hinders the rational design of stapled peptides as PPI inhibitors. Using the guanylate kinase (GK) domain of the postsynaptic density protein 95 (PSD-95) as the target, we quantified the enthalpic and entropic contributions by combining isothermal titration calorimetry (ITC), X-ray crystallography, and free energy calculations based on all-atom molecular dynamics (MD) simulations. We successfully designed a stapled peptide inhibitor (staple 1) of the PSD-95 GK domain that led to a 25-fold increase in the binding affinity (from tens of μMs to 1.36 μM) with high cell permeability. We showed that entropy indeed greatly enhanced the binding affinity and the entropy gain was mainly due to the constrained-helix structure of the stapled peptide in solution (free state). Based on staple 1, we further designed two other stapled peptides (staple 2 and 3), which exerted even larger entropy gains compared to staple 1 because of their more flexible bound complexes (bound state). However, for staple 2 and 3, the overall binding affinities were not improved, as the loose binding in their bound states led to an enthalpic loss that largely compensated the excess entropy gain. Our work suggests that increasing the stability of the stapled peptide in free solution is an effective strategy for the rational design of stapled peptides as PPI inhibitors.

Characterizing the Binding Sites for GK Domain of DLG1 and DLG4 via Molecular Dynamics Simulation.

Discs-large (DLG) is a member that belongs to the membrane-associated guanylate kinase (MAGUK) family. The GK domain of DLGs has evolved into a protein–protein interaction module that could bind with kinds of proteins to regulate diverse cellular functions. Previous reports have demonstrated the GK domain of DLGs functioned as a phosphor-peptide-binding module by resolving the crystal structures. Here we investigated into the interactions of DLG1 and DLG4 with their reported phosphor-peptides by molecular dynamics simulations. Post-dynamics analysis showed that DLG1/4 formed extensive interactions with phosphorylated ligands, including hydrophobic and hydrogen bonding interactions. Among them, the highly conserved residues among the DLGs in phosphor-site and β5 sheet were crucial for the binding according to the energy decomposition calculations. Additionally, the binding interactions between DLG4 and reported unphosphorylated peptides including MAP1A and designed GK inhibitory (GKI-QSF) peptides were analyzed. We found the key residues that played important roles in DLG4/unphosphorylated peptide systems were very similar as in DLG4/phosphor-peptide systems. Moreover, the molecular dynamic simulation for the complex of DLG1 and GKI-QSF was carried out and predicted that the GKI-QSF could bind with DLG1 with similar Kd value compared to DLG4/GKI-QSF, which was verified by using ITC assay (Kd = 1.20 ± 0.29 μM). Our study might be helpful for the better understanding of the structural and biological function of DLGs GK domain and encourage the discovery of new binders.

Identification and Visualization of Functionally Important Domains and Residues in Herpes Simplex Virus Glycoprotein K(gK) Using a Combination of Phylogenetics and Protein Modeling

Alphaherpesviruses are a subfamily of herpesviruses that include the significant human pathogens herpes simplex viruses (HSV) and varicella zoster virus (VZV). Glycoprotein K (gK), conserved in all alphaherpesviruses, is a multi-membrane spanning virion glycoprotein essential for virus entry into neuronal axons, virion assembly, and pathogenesis. Despite these critical functions, little is known about which gK domains and residues are most important for maintaining these functions across all alphaherpesviruses. Herein, we employed phylogenetic and structural analyses including the use of a novel model for evolutionary rate variation across residues to predict conserved gK functional domains. We found marked heterogeneity in the evolutionary rate at the level of both individual residues and domains, presumably as a result of varying selective constraints. To clarify the potential role of conserved sequence features, we predicted the structures of several gK orthologs. Congruent with our phylogenetic analysis, slowly evolving residues were identified at potentially structurally significant positions across domains. We found that using a quantitative measure of amino acid rate variation combined with molecular modeling we were able to identify amino acids predicted to be critical for gK protein structure/function. This analysis yields targets for the design of anti-herpesvirus therapeutic strategies across all alphaherpesvirus species that would be absent from more traditional analyses of conservation.

Intramolecular domain dynamics regulate synaptic MAGUK protein interactions.

PSD-95 MAGUK family scaffold proteins are multi-domain organisers of synaptic transmission that contain three PDZ domains followed by an SH3-GK domain tandem. This domain architecture allows coordinated assembly of protein complexes composed of neurotransmitter receptors, synaptic adhesion molecules and downstream signalling effectors. Here we show that binding of monomeric CRIPT-derived PDZ3 ligands to the third PDZ domain of PSD-95 induces functional changes in the intramolecular SH3-GK domain assembly that influence subsequent homotypic and heterotypic complex formation. We identify PSD-95 interactors that differentially bind to the SH3-GK domain tandem depending on its conformational state. Among these interactors, we further establish the heterotrimeric G protein subunit Gnb5 as a PSD-95 complex partner at dendritic spines of rat hippocampal neurons. The PSD-95 GK domain binds to Gnb5, and this interaction is triggered by CRIPT-derived PDZ3 ligands binding to the third PDZ domain of PSD-95, unraveling a hierarchical binding mechanism of PSD-95 complex formation.

Molecular Mechanism of Voltage-Gated Ca2+ Channel Regulation by Membrane PIP2

Voltage-gated calcium (CaV) channels play essential roles in adjusting calcium influx upon membrane depolarization. CaV2 (N-, P/Q- and R-type) channels are concentrated in the presynaptic nerve terminals and important for the neurotransmitter release. Adjusting the presynaptic calcium channel gating exerts potent influence on synaptic plasticity. CaV channels need auxiliary subunits for proper trafficking to the plasma membrane and the channel gating. Especially CaVβ subunit plays crucial roles in the surface expression of CaV channels and fine-tuning of channel gating. It has been known that CaV channels are modulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP2). The binding affinity between ion channel and PIP2 is important for the channel gating normally, but molecular mechanism of PIP2 regulation remains unclear. It was recently reported that subcellular localization of β subunit is a key factor for the control of PIP2 sensitivity of CaV channels. Here we found that the intracellular movement of I-II linker in a1 subunit is important for determining the PIP2 sensitivity of CaV channels. When the I-II linker was shifted to the plasma membrane, current inhibition by PIP2 depletion significantly decreased as like the responses triggered by membrane-tethered β subunit. Consistently we also found that inserting a flexible linker between membrane-tethered Lyn and GK domain of β subunit increased the PIP2 sensitivity of CaV channels. Polybasic motif at the C-terminal end of the I-II linker of CaV channels is a potential PIP2 interaction site. Neutralization of the polybasic motif of I-II linker abolished PIP2 sensitivity of CaV channels. Together, our results indicate that the conformational shift of I-II linker to the plasma membrane is the key mechanism for decreasing the PIP2 sensitivity of CaV channels and this shift is mainly regulated by auxiliary CaVβ subunit in physiological condition.

ELKS/Voltage-Dependent Ca2+ Channel-β Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic β Cells

Pancreatic β cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, isused in β cells to regulate Ca2+ influx for insulin secretion. β cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of β cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the β cell plasma membrane, which were markedly decreased in ELKS-KO β cells. Mechanistically, ELKS directly interacted with the VDCC-β subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the β cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.

Postsynaptic localization and regulation of AMPA receptors and Cav1.2 by β2 adrenergic receptor/PKA and Ca2+/CaMKII signaling.

The synapse transmits, processes, and stores data within its tiny space. Effective and specific signaling requires precise alignment of the relevant components. This review examines current insights into mechanisms of AMPAR and NMDAR localization by PSD-95 and their spatial distribution at postsynaptic sites to illuminate the structural and functional framework of postsynaptic signaling. It subsequently delineates how β2 adrenergic receptor (β2 AR) signaling via adenylyl cyclase and the cAMP-dependent protein kinase PKA is organized within nanodomains. Here, we discuss targeting of β2 AR, adenylyl cyclase, and PKA to defined signaling complexes at postsynaptic sites, i.e., AMPARs and the L-type Ca2+ channel Cav1.2, and other subcellular surface localizations, the role of Akinase anchor proteins, the physiological relevance of the spatial restriction of corresponding signaling, and their interplay with signal transduction by the Ca2+- and calmodulin-dependent kinase CaMKII How localized and specific signaling by cAMP occurs is a central cellular question. The dendritic spine constitutes an ideal paradigm for elucidating the dimensions of spatially restricted signaling because of their small size and defined protein composition.

Activation of Discs large by aPKC aligns the mitotic spindle to the polarity axis during asymmetric cell division.

Asymmetric division generates cellular diversity by producing daughter cells with different fates. In animals, the mitotic spindle aligns with Par complex polarized fate determinants, ensuring that fate determinant cortical domains are bisected by the cleavage furrow. Here, we investigate the mechanisms that couple spindle orientation to polarity during asymmetric cell division of Drosophila neuroblasts. We find that the tumor suppressor Discs large (Dlg) links the Par complex component atypical Protein Kinase C (aPKC) to the essential spindle orientation factor GukHolder (GukH). Dlg is autoinhibited by an intramolecular interaction between its SH3 and GK domains, preventing Dlg interaction with GukH at cortical sites lacking aPKC. When co-localized with aPKC, Dlg is phosphorylated in its SH3 domain which disrupts autoinhibition and allows GukH recruitment by the GK domain. Our work establishes a molecular connection between the polarity and spindle orientation machineries during asymmetric cell division.

CHARACTERIZATION OF THE CASK PROTEIN IN DROSOPHILA OVARIES

CASK (Ca2+/Calmodulin Serine Threonine Kinase) is a member of the MAGUK family of proteins. The protein contains the canonical MAGUK domains (PDZ domain, the GK domain and the SH3 domain) as well as an N‐terminal CamK‐like domain. CASK and other MAGUK family proteins, including Dlg, have been shown to function in the establishment and maintenance of cell polarity in a variety of cell types. Interestingly, a recent screen has identified CASK as regulator of border cell migration, a model of collective cell migration that occurs during Drosophila oogenesis. Border cell migration occurs during Stage 9 of Drosophila egg development and is a process by which two non‐motile polar cells located at the anterior of the egg chamber signal 4–8 adjacent follicular epithelial cells to transform into motile border cells. Once signaled, the border cells will transport the polar cells to the anterior end of the oocyte within the egg chamber where they will form the micropyle. The movement of this border cell cluster to the anterior end of the oocyte is completed by stage 10 of Drosophila ovary development. Despite the putative role for CASK in border cell migration, very little work has been done to characterize CASK's function in the Drosophila ovaries. The current study aims to begin to characterize CASK's basic gene expression in Drosophila ovaries and optimize a protocol for immunohistochemistry such that border cell migration assays can be conducted on dissected stage 10 egg chambers. RT‐PCR analysis was conducted with primers specific to each of the seven CASK isoforms known to be present in Drosophila (CASK‐A, ‐B,‐D, ‐E, ‐F, ‐G, ‐H). Thus far expression of the CASK‐A, CASK‐B, CASK‐H and CASK‐E isoforms have been confirmed in Drosophila ovaries. Studies were also conducted to determine the optimal time in which the flies should be fed yeast to yield the highest percentage of stage 10 ovaries for subsequent border cell migration assays. Feeding flies a wet yeast paste is known to provide a nutritive enhancement for female oogenesis. Yeast time trials were conducted in which 3 lines of wild‐type flies and the p18 CASK knockout fly line were allowed to feed on yeast for 12, 13 or 14 hours. Results indicated that the 13‐hour yeasting time yielded the highest number of stage 10 ovaries. This time was consistent across genotypes. Future work will continue to assess CASK gene expression via RT‐PCR with primers specific to the CASK‐D, CASK‐F and CASK‐G isoforms. Defects in border cell migration will also be assessed via immunohistochemical analysis with the singed antibody on Drosophila utilizing the yeast time trial results attained in this study. Characterization of CASK's gene expression and assays of border cell migration in CASK knockout flies will serve to elucidate the molecular mechanisms by which CASK contributes to group cell movement. Furthermore, this work will also contribute to a greater understanding of the types of proteins required for mediating cell migration during biological processes such as wound healing, inflammation and tumor cell metastasis.

Deletion of a Predicted β-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons.

We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31-68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31-68 mutation spans a well-defined β-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31-68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31-68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses. Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted β-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our previous observations that deletion of this gK domain prevents the virus from successfully infecting ganglionic neurons after ocular infection of mice.

A Low Affinity Cavb4a/Rem2 Interaction is Required for Inhibition of Cav2.1 Ca2+ Currents

RGK (Rem, Rad, Kir/Gem proteins) proteins, including Rem2, cause profound inhibition of high‐voltage activated Ca2+channels containing intracellular regulatory b subunits. All RGK proteins bind to Cavb subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies solution state nuclear magnetic resonance (NMR) and calorimetric techniques to map the binding site for Rem2 on human Cavb4a and to measure the binding affinity. Our experiments revealed two binding surfaces on the b4 GK domain contributing to a 156 μM Kd interaction: 1) a hydrophobic pocket surrounded by four residues, mutation of any of which completely disrupted binding; and 2) a nearby surface containing three residues that when individually mutated decreased affinity. Cav2.1 Ca2+currents were completely inhibited by Rem2 when co‐expressed with wild‐type Cavb4a, but were unaffected by Rem2 when co‐expressed with a Cavb4a site 1 mutant. These results provide direct evidence for a low affinity Rem2/Cavb4 interaction and its essential role in Cav2.1 inhibition by Rem2.

Motor Impairments, Striatal Degeneration, and Altered Dopamine-Glutamate Interplay in Mice Lacking PSD-95

Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32–positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor–mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95–dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.

Structural and Binding Studies of the Cav1.1 β1A Subunit

Excitation-contraction (EC) coupling in skeletal muscle requires a physical coupling between the voltage-gated calcium channel (Cav1.1) in the surface membrane and the skeletal ryanodine receptor (RyR1) Ca2+ release channel in the membrane of the sarcoplasmic reticulum Ca2+ store. Although the exact molecular mechanism of EC coupling is unresolved, both the α1s and β1a subunits of Cav1.1 are essential for this process. The β1a subunit has a modular structure consisting of SH3/guanylate kinase (GK) domains separated by a variable hook region. The GK domain binds with high affinity to the I-II loop of the α1 subunit, but the functional significance of the SH3 domain remains undefined.Until now the structure of the Cav1.1 β1a subunit has not been experimentally determined, but other Cav β-isoform structures have suggested that the SH3 binding site is occluded, preventing binding to polyproline-rich partners. This prediction is at odds with our findings that show the Cav1.1 β1a subunit and the α1s subunit II-III loop interact (Kd = ∼3 μM). We demonstrate that this interaction takes place through the SH3 domain of the β1a subunit and a proline-rich region of the α1s II-III loop, which has previously been shown to be critical for skeletal-type EC-coupling (1). Through mutational studies we demonstrate that isoform-specific differences in the SH3 RT loop enable the interaction of the β1a SH3 domain with proline-rich binding motifs.Our determination of the crystal structure of Cav1.1 β1a provides the first opportunity to examine differences between this isoform and other published structures. In light of this novel structure and binding data, we discuss the specific role of the β1a subunit in EC coupling and its relationship with the Cav1.1 α1 subunit and RyR1.1. Kugler, G. et al (2004). J Biol Chem 279(6): 4721-4728.

Characterization of the first honeybee Ca2+ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation

The honeybee is a model system to study learning and memory, and Ca(2+) signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca(2+) channel subunit. We identified two splice variants of the Apis CaVβ Ca(2+) channel subunit (Am-CaVβ) and demonstrated expression in muscle and neurons. Although AmCaVβ shares with vertebrate CaVβ subunits the SH3 and GK domains, it beholds a unique N terminus that is alternatively spliced in the first exon to produce a long (a) and short (b) variant. When expressed with the CaV2 channels both, AmCaVβa and AmCaVβb, increase current amplitude, shift the voltage-sensitivity of the channel, and slow channel inactivation as the vertebrate CaVβ2a subunit does. However, as opposed to CaVβ2a, slow inactivation induced by Am-CaVβa was insensitive to palmitoylation but displayed a unique PI3K sensitivity. Inactivation produced by the b variant was PI3K-insensitive but staurosporine/H89-sensitive. Deletion of the first exon suppressed the sensitivity to PI3K inhibitors, staurosporine, or H89. Recording of Ba(2+) currents in Apis neurons or muscle cells evidenced a sensitivity to PI3K inhibitors and H89, suggesting that both AmCaVβ variants may be important to couple cell signaling to Ca(2+) entry in vivo. Functional interactions with phospho-inositide and identification of phosphorylation sites in AmCaVβa and AmCaVβb N termini, respectively, suggest that AmCaVβ splicing promoted two novel and alternative modes of regulation of channel activity with specific signaling pathways. This is the first description of a splicing-dependent kinase switch in the regulation of Ca(2+) channel activity by CaVβ subunit.

Functional Analysis of the C-Terminal Region of γ-Glutamyl Kinase ofSaccharomyces cerevisiae

γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.